Background: Regular physical activity (PA) and reduced sedentary behavior (SB) are positively related to children's health and considered as pillars of a healthy lifestyle. Full-day schools with their afterschool programs (ASPs) have an impact on children's daily PA and SB. Studies investigating PA and SB in ASPs, which compare PA and SB between the organizational forms full-day and half-day schools, are rare. The aim of this study is to describe elementary school children's PA and SB during ASPs and to compare the results to other time periods of the day, e.g., teaching hours and leisure time. Additionally, PA and SB of children in full-day and half-day schools are compared. Further, relevant factors influencing the achievement of the World Health Organization's (WHO's) PA guidelines for children, e.g., time spent in ASPs, are investigated.Methods: PA and SB of 332 German students  from 11 different elementary schools were measured via accelerometry for 5 consecutive days within one school week in 2017. PA and SB during ASPs and other times of the day were analyzed via one-way and factorial ANOVA, correlation, and logistic regression.Results: Children attending full-day schools show the highest percentage of moderate-to-vigorous PA (MVPA) (13.7%) and the lowest percentage of SB (49.5%) during ASPs, in comparison with teaching hours and leisure time. In the afternoon hours, full-day school children show 20 min less SB than half-day school children. Children spending more time in ASPs obtain significantly more SB (r = 0.23) and less MVPA (r = −0.15). Further, they less likely reach WHO's PA guidelines odds ratio (OR = 0.98).Conclusion: Peers and the choice as well as offer of extracurricular activities promote PA in ASPs. Media availability leads to higher SB in leisure time. ASPs help to be more active and less sedentary. Time spent in ASPs should be limited, so that full-day school children still have the possibility to join other PA offers in leisure time. ASP time should contain a certain minimum amount of MVPA in line with ASP guidelines. Social Determinants of Health, behavior patterns such as PA and SB are significantly influenced by the social network and living as well as working conditions. For children, these living and working conditions are primarily the family, the commune they grow up in, and the school they attend. Considering the amount of time children spend at school on weekdays, the factor school contributes substantially to children's daily PA as well as SB and therefore their health.Physical inactivity and sedentary behavior (SB) are determinants of poor health . On the In industrialized countries, almost every child is enrolled in school, and school has the most intensive and continuous impact on children aged 6–15 . In the Global Recommendations on Physical Activity for Health (WHO PA GL) are a marker for a healthy lifestyle in the meaning of daily PA. Besides the WHO PA GL, there are several recommendations, policies, or guidelines regarding the amount of PA in ASPs  recommends at least 60 min of moderate-to-vigorous PA (MVPA) daily for children aged 5–17 . These G in ASPs . These r in ASPs –15. It i in ASPs . Most or in ASPs . This me in ASPs . The WHOromotion  recommenromotion –15. Screromotion . The Calromotion .Several studies investigated PA and SB in the school context, especially considering full-day school's ASPs: Beets et al.  showed iOverall, most studies in this field analyzed children's daily PA and SB, without differentiating between times of the day. Research analyzing PA and SB in ASPs and its potential influence on daily PA as well as studies comparing full-day school and half-day school are rudimentary. Studies investigating PA and SB in German full-day schools accurately and with device-based measurements, such as accelerometry, are missing. Only one international study  comparedthe description of PA and SB of children attending full-day schools during time spent in ASPs, and additionally, the comparison of PA and SB in ASPs with PA and SB during teaching hours and leisure time;the comparison of full-day school and half-day school children's PA and SB during the afternoon hours; andthe investigation of the impact of full-day school specific factors on the achievement of WHO PA GL.Physical Activity in Full-Day School Children funded by the German Federal Ministry of Education, Youth and Sports Baden Württemberg from 2014 to 2018. The study was performed from May to July 2017 in elementary schools with either a mandatory or voluntary full-day school branch located in the federal state of Baden-Württemberg, Germany. To minimize weather condition-related differences in PA and SB, data were collected in spring and summer. Regional weather conditions  were documented and checked using data from the German Weather Service.This study was part of the project In the first step, 25 schools—officially registered, accredited, and therefore supported by the Federal Ministry of Education, Youth and Sports Baden-Württemberg in their first year as full-day school—were asked to join the study. Eleven schools (44%) agreed to participate. Overall, 1,620 children from grade 1 to 4 were enrolled in the 11 participating schools. In the second step, principals and class teachers handed out an information letter about the study. Parents were asked to fill out the included reply form to accept or decline their children's participation in the study. Regardless of their affiliation to half-day or full-day school, all children of grade 1 to grade 4 were invited to participate. Data were collected by accelerometry for device-based measurement of PA and SB and by paper–pencil questionnaire .n = 10) and lack of wear time (n = 186), the final sample included 332 children with adequate data for 5 consecutive school days . Because of broken devices  at the left hip with an elastic belt. Robusto and Trost's  study hiData collection by accelerometer was set to 10-s epoch length and started 1–2 h after distribution to avoid increased PA results because of curiosity for device and study. ActiLife Software v6.11.9 was used for data processing. Wear time validation was calculated using Troiano  defaultsMotorik-Modul activity questionnaire MoMo-AFB (Socio-demographic data (date of birth and sex) were collected via paper–pencil questionnaire, which was an adapted version of the MoMo-AFB . Childrenon-teaching time was created. Children attending full-day schools stay in school for extracurricular activities in ASPs. Leisure time with activities free of choice started after ASPs. Non-teaching time for full-day school children included ASPs and leisure time. Children in half-day schools left school after teaching hours. The remaining time awake was leisure time and therefore equal to non-teaching time. Valid wear time was checked for full day recording (as mentioned above) and double-checked for each period of the day to ensure at least 50% wear time in each time period. On average, daily wear time amounted to 11.92 (SD = 2.3) hours and 49.7% (SD = 9.5) for a whole day; 4.06 (SD = 0.7) hours in teaching hours, which equates to 89.3% (SD = 12.8) for daily teaching hours, 2.46 (SD = 1.0) hours and 91.6% (SD = 14.4) in ASPs, 6.72 (SD = 2.1) hours and 50.6% (SD = 13.5) in leisure time, and 7.75 (SD = 1.9) hours and 54.1% (SD = 12.5) in non-teaching time.PA and SB data were divided into different time periods throughout the day and summarized for 5 consecutive school days from Monday to Friday. Individual timetables were created, based on class schedule and ASP attendance information. A typical school day was divided into teaching hours and leisure time for half-day school children and teaching hours, ASPs, and leisure time for full-day school children. To compare PA and SB in the afternoon hours between full-day and half-day school children, an additional time period named After raw data to PA and SB data with ActiLife® software were converted, Microsoft® Excel® for Office 365 MSO was used to prepare data.post-hoc test was used. Effect size eta squared was categorized based on Cohen  and specific full-day school predictors  as independent variables, which were given as odds ratio (OR) to achieve the WHO PA GL (dependent variable). Significance was assessed at α = 0.05 for all analyses. All statistical tests were conducted using IBM® SPSS® Statistics (version 26).Comparisons of children's PA and SB in different time periods  within the day were conducted by repeated measure factorial ANOVA with sex and grade as independent variables. Analyses comparing full-day and half-day school children or subgroups differentiated by school affiliation were fulfilled by one-way ANOVA, to identify the interaction effect between sex and grade, a factorial ANOVA was conducted. For multiple comparisons, Tukey's on Cohen : small, n = 198) and taking part in ASPs on at least 1 and up to 5 days of the week. Over two thirds of students attending full-day school participate on three (31.8%) or four (37.4%) afternoons in ASPs. The number of days varies in the full range from one (6.6%) or two (7.1%), up to five (17.2%) days attending full-day school programs within a school week with an average of 3.52 (SD = 1.1) days weekly. On average, children spend 162.88 (SD = 57.0) minutes per day in ASPs. The maximum duration of ASPs time is 5 h/day. Summarizing all school days within 1 week, we found that children attend extracurricular activities in ASPs for 9 h 42 min  on average.The following results refer to the subgroup of participants attending full-day school  = 1.32, p = 0.263, η2 = 0.03] in percentage of SB. Both factors individually show significant differences: boys' SB is lower than girls' . Differentiated by grade, there is a significant difference of SB in ASPs . Tukey's post-hoc multiple comparisons show that only first graders show significantly less SB than do second, third, and fourth graders. Considering the percentage of time spent in MVPA during ASPs, only sex shows a significant difference . There are neither significant differences between grades  nor an interaction effect . Significant differences with large effects are found for school affiliation in both percentage in SB  and MVPA . Post-hoc results reveal that only one school stands out with a highly active ASP. This school is the only one in the sample where full-day school is mandatory for all children. PA and SB data of this school (n = 27) show that children spend on average 18.4% (SD = 8.2) of ASPs time in MVPA , which differs significantly in comparison with four schools in a multiple comparison. In 39.0% (SD = 10.0) of ASP time, children of this school are sedentary , which is significantly different to eight schools.r = 0.23, p = 0.001) and a slightly negative correlation for MVPA percentages  as seen in Children attending full-day school stay on average 2 h 43 min  per school day at school for extracurricular activities in ASPs. Regarding the duration, there is a small positive correlation for SB  time is significantly the most active and least sedentary time of the day than are teaching hours  and leisure time  with the highest percentage of MVPA and lowest percentage of SB. Considering SB, interaction effects of the repeated measure factorial ANOVA are significant for the time periods differentiated by sex, but not by grade or by sex and grade. For percentages of MVPA, significant interaction effects can be found for sex as well as grade, but not for grade and sex see , 3. Descnon-teaching time children attending full-day schools  spend significantly less minutes in SB than children in half-day schools : F = 4.67, p = 0.031, η2 = 0.01. The difference in minutes of MVPA in the non-teaching time is not significant; half-day school children  spend 1.7 min less in MVPA than children attending full-day schools : F = 0.63, p = 0.427, η2 < 0.01. Considering girls and boys separately, the affiliation to school's organizational forms do not differ in boys' SB [F = 0.33, p = 0.570, η2 < 0.01) or MVPA [F = 0.33, p = 0.565, η2 < 0.01). The difference in minutes of SB in the non-teaching time for girls aggregates to almost 30 min. Girls in half-day schools show SB for 290.47 (SD = 87.19) minutes and girls in full-day schools for 260.73 (SD = 85.29) min [F = 5.02, p = 0.026, η2 = 0.03). As reported for boys' PA, there are no significant differences in girls' MVPA [F = 0.06, p = 0.801, η2 < 0.01). Regarding subgroups divided by grade, there is a difference in second graders: half-day school children in second grade spend significantly more time in SB during non-teaching time than full-day school children [F = 5.75, p = 0.019, η2 = 0.06). For all other grades, differences in SB and PA between full-day and half-day school children are not significant.During 2(1) = 24.53, p < 0.001, φ = 0.27). Attendance to full-day or half-day school does not show any significant difference in the percentage of children reaching the WHO PA GL: full-day school 62.1%, half-day school 56.7% (χ2(1) = 0.97, p = 0.324, φ = 0.05).In this study, 59.9% of participants reach the recommended 60 min of MVPA daily according to the WHO PA GL . A chi-sThe logistic regression model with six predictors reaches a likelihood of 79.1%. The OR is displayed in National Afterschool Association  help to identify specific subgroups or (b) highlight the school's organizational form influence on children's PA and SB. Compared with sports clubs, full-day schools with optional sports activities reach more children. A potential influence of socioeconomic issues on children's sports activities may be much smaller in full-day schools' ASPs than in organized activities in sports clubs. Additionally, activities in ASPs could have a recruitment effect on club sports .Findings of this study may lead to the following practical implications: time in ASPs should be limited in duration and not exceed the late afternoon. Full-day schools should offer a variety of extracurricular activities in ASPs for the time children stay in school. Children attending full-day school should still have the possibility to participate in organized sports, e.g., organized by sports clubs. Therefore, full-day schools and sports clubs should cooperate. Because of a lack of teachers, providing extracurricular activities for the whole time in ASPs is a problem for many schools. Including sports club coaches in ASPs may help to offer more activities during ASPs and at the same time enable sports clubs' recruitment of interested children to participate in organized sports. The compliance of PA-based ASP guidelines may help to lead children not engaged in organized sports to more PA and less SB.N = 332 distributed over 11 elementary schools. Accelerometer-based PA and SB data underlies the chosen algorithms to calculate wear time and cut points. Algorithms used in this study were chosen by best fitting arguments. Comparisons with studies using other algorithms should be done carefully  may be attributed to the considerable effort for parents, managing questionnaire completion, and accelerometer recording for a whole week. Although parents were informed about privacy and the Federal Ministry's approval of the study, data collection via accelerometry might still be seen critically by some parents.A strength of this study is the device-based measurement of PA and SB via accelerometry with a sample size of arefully . Althougarefully  as well The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Commitee of the University of Konstanz . Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.AK and FM coordinated the whole project and developed the study design. AK collected data and wrote the paper with substantial contributions from CM and MS. AK and CM performed the data preparation and statistical analysis. MS performed the final proofreading. All authors provided feedback on drafts and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ACVR1A/ALK2 gene. Activin-A is a key molecule for initiating the process of HO via the activation of mTOR, while rapamycin, an mTOR inhibitor, effectively inhibits the Activin-A-induced HO. However, few reports have verified the effect of rapamycin on FOP in clinical perspectives.Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal-dominant disease characterized by heterotopic ossification (HO) in soft tissues and caused by a mutation of the We investigated the effect of rapamycin for different clinical situations by using mice conditionally expressing human mutant ACVR1A/ALK2 gene. We also compared the effect of rapamycin between early and episode-initiated treatments for each situation.Continuous, episode-independent administration of rapamycin reduced the incidence and severity of HO in the natural course of FOP mice. Pinch-injury induced HO not only at the injured sites, but also in the contralateral limbs and provoked a prolonged production of Activin-A in inflammatory cells. Although both early and injury-initiated treatment of rapamycin suppressed HO in the injured sites, the former was more effective at preventing HO in the contralateral limbs. Rapamycin was also effective at reducing the volume of recurrent HO after the surgical resection of injury-induced HO, for which the early treatment was more effective.Our study suggested that prophylactic treatment will be a choice of method for the clinical application of rapamycin for FOP. Fibrodysplasia ossificans progressiva (FOP) OMIM#135100) is a rare autosomal dominant hereditary disease characterized by congenital malformations of the great toes and progressive heterotopic ossification (HO) in fibrous tissues such as skeletal muscles, tendons, and ligaments. The prevalence has been estimated at approximately one in 2 million people 35100 is . CorticoACVR1A/ALK2 (activin receptor 1A/activin-like kinase-2), which encodes a type I receptor for bone morphogenetic protein (BMP) [The disease-causing gene of FOP is in (BMP) . More thin (BMP) . Histoloin (BMP) . Althougin (BMP)  and rescin (BMP) . We induin (BMP) . Around in (BMP) .Activin-A is a member of the TGFβ protein family and transduces TGFβ signaling by binding to ACVR1B/ALK4 or ACVR1C/ALK7 . HoweverAlthough FOP is progressive, the clinical course is not a simple time-dependent course, but rather a step-wise progression –4. It haACVR1A/ALK2 gene.To design the appropriate schedule for administration, here we investigated the effect of rapamycin for FOP by mimicking non-flare-up, flare-up-induced, and surgery-induced HO in mice that conditionally express human mutant The establishment of FOP-ACVR1 conditional transgenic mice  was reported previously , and 13-Rapamycin was purchased from MidChem Express and dissolved in 10% DMSO with 0.5% w/v methylcellulose 400. Rapamycin was injected intraperitoneally at 5 mg/kg once a day, 5 times a week. Mice in the vehicle-treated group received the same solution without rapamycin. All mouse experiments and analyses of the results were performed under blinded condition.Under isoflurane anesthesia  , a skin incision (approximately 1.5 cm) was made on the lateral surface of the left hindlimb, and the left gastrocnemius muscle was exposed. The middle portion of the muscle was pinched by a tissue forceps for 5 sec, and then the skin was closed.Twenty-one days after the pinch-injury, the injured sites were exposed under anesthesia, and ossified tissues were resected with a high-speed drill and bipolar electrocautery under loupe magnification as much as possible.For X-ray and μCT imaging, mice were anesthetized with a mixture of medetomidine, midazolam, and butorphanol. X-ray images were scanned using DX-50 . μCT images were scanned using an X-ray CT system  and analyzed with TRI/3D-BON software  according to the manufacturer’s instructions. Investigation was completed under a blinded condition of the experimental groups.Collected tissue samples were fixed with 4% paraformaldehyde for 48 h, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E), Safranin O, and von Kossa.2, the spleens were extirpated and homogenized using a Multi-Beads Shocker  according to the manufacturer’s instructions. Total RNA was extracted from homogenized tissues using Sepasol-RNA I Super G  and the RNeasy Kit  and treated with the DNase-One Kit (QIAGEN) to remove genomic DNA. cDNA was synthesized using ReverTra Ace qPCR RT Master Mix . Quantitative PCR (qPCR) was performed with Thunderbird SYBR qPCR Mix (TOYOBO) and analyzed with the QuantiStudio 12 K Flex Real-time PCR System . The primer sequences are described in Additional file Gapdh.After euthanizing the mice by COp < 0.05 or **p < 0.01.Statistical analysis was performed using JMP Pro® 14 . The statistical significance was calculated using the Student’s t-test, Dunnett test, or Wilcoxon sum rank test, as described in the corresponding figure legends. Survival was assessed by log-rank. Statistical significance is indicated as *n = 9) or rapamycin-treated group (n = 10)  group, in which DOX administration was started 14 days before the pinch-injury, and DOX (−) group, in which a vehicle was administrated. Mice were sacrificed at 3, 7, and 14 days after the injury, X-ray photos of the lower limbs were taken, and muscle samples were obtained from the injured sites at each time point Fig. a and b.The histology of the skeletal muscle before the pinch-injury did not show a clear difference between the DOX (+) and DOX (−) groups Fig. a, Day 0.These results are compatible with previous data of HO in FOP patients , indicatFOP-ACVR1A in spleen cells and interestingly inhibited endogenous ACVR1A gene expression /Pinch (+) mice than in the other groups even 21 days after the injury  group, but not in the Dox (−) group (Additional file To investigate the systemic effect after the pinch-injury and HO in FOP-ACVR1 mice, particularly for inflammatory cells that are known to produce Activin-A , we analion Fig. d. It is We next examined the effect of rapamycin on pinch-injury-induced HO Fig. a. Seven All mice received the pinch-injury at Day 14 and were sacrificed at Day 35 (Fig. We investigated the new HO in the contralateral limbs and found that it was formed in all mice in the control group Fig. d. The inSupposing that the inflammation at the injured site was responsible for the HO in the contralateral side, we evaluated the effect of rapamycin on the inflammatory stage of HO in FOP-ACVR1 mice. Prior to the pinch-injury, mice in the control group were treated with vehicle, and those in the early treatment group were treated with rapamycin for 7 days and sacrificed 3 days after the pinch-injury (Additional file Finally, we evaluated the effect of rapamycin after the surgical removal of HO Fig. a. Pinch-Surgical resection evokes much stronger systemic inflammation than pinch-injury and therefore may propagate systemic HO. Notably, two mice in each group showed new HO in the contralateral limbs after pinch-injury Fig. e.In summary, both early and episode-initiated treatments are effective at suppressing episode-induced HO, but early treatment seemed more effective at preventing the systemic propagation of HO, as evidenced by the observations of contralateral HO.The identification of Activin-A as the initiating factor has disclosed the molecular process of HO in FOP, enabling the investigation of stage-specific targets and treatments. The first step of HO is the production of Activin-A by locally infiltrating inflammatory cells such as monocytes, macrophages, and mast cells, which can produce Activin-A by exogeneous stimuli and were reported to be abundant in the inflammatory phase of HO . The genAs more drugs come to the stage of clinical trial, each drug may have a suitable time-window for exerting its optimal effect. The inhibitory effect of rapamycin on HO was initially shown to act through the inhibition of HIF-1-α, which is one of the downstream targets of mTOR . HIF-1-αIt should be considered, however, that the continuous administration of rapamycin will exert adverse effects. As an immunosuppressant after kidney transplantation, rapamycin has been used for growing children for long periods, and some clinical studies reported that the long-term use of rapamycin reduced their growth velocity and induced a smaller height , 28. ExpIt should be also noticed that the inhibitory effect of early treatment for the recurrence after the surgical resection was limited and may be insufficient in the clinical situation. Surgical resection may damage muscle tissues more gravely with massive bleeding than the pinch-injury, and thus may provoke much stronger inflammatory signals and recruit a larger number of precursor cells at the resected sites. The administration of a temporally high amount of rapamycin with or without other anti-inflammatory reagents should therefore be considered for future application.The experimental treatments demonstrated in this study, which were designed based on the clinical situations of FOP patients, will contribute to the exploration of effective treatment methods using rapamycin and also other candidate drugs for improving the quality of life of FOP patients.Additional file 1:Table S1. Information of primers used in qRT-PCRAdditional file 2:Figure S1. Total bone volume and body weight of mice in each groupAdditional file 3:Figure S2. Serum level of cytokines after pinch-injuryAdditional file 4:Figure S3. Representative histological findings of the inflammatory stage of HO.Additional file 5:Figure S4. Representative histological findings of recurrent HOAdditional file 6. Supplementary methods


Immunoscore have shown a promising prognostic value in many cancers. We aimed to establish and validate an immune classifier to predict survival after curative resection of hepatocellular carcinoma (HCC) patients who have undergone curative resection.The immunohistochemistry (IHC) classifier assay was performed on 664 patients with Barcelona Clinic Liver Cancer (BCLC) stage 0 or A HCC. A nine-feature-based HCC-IHC classifier was then constructed by the least absolute shrinkage and selection operator method. The associations between the HCC-IHC classifier and patient outcomes were assessed. Herein, a nomogram was generated from the Cox regression coefficients and evaluated by decision curve analysis.P < 0.001). The HCC-IHC classifier-based nomogram presented better accuracy than traditional staging systems.We constructed an HCC-IHC classifier based on nine features; significant differences were found between the low-HCC-IHC classifier patients and high-HCC-IHC classifier patients in the training cohort in the 5-year relapse-free survival rates (46.7% vs. 26.7%, respectively; In conclusion, the HCC-IHC classifier could effectively predict recurrence in early-stage HCC patients and supplemented the prognostic value of the BCLC staging system. The HCC-IHC classifier may facilitate patient decision-making and individualise the management of postoperative patients with early-stage HCC. Given their moderate prediction accuracy, new strategies to stratify cancer patients that have focussed on tumour cell-, tumour mutational burden-, signalling pathway-, and gene expression-based classifications are of significant usefulness,7 new strategies focussing on tumour microenvironment are also urgently needed for the advancement of clinical outcome prediction to supplement the current staging system.8Although imperative developments have been made in the early diagnosis of hepatocellular carcinoma (HCC), there has not been a relative advance in the prediction of patient recurrence.11 In HCC, we have shown that innate and adaptive immune components, such as regulatory T cells (Tregs), macrophages, and neutrophils, in the tumour and invasive margin were strongly correlated with overall survival (OS) and relapse-free survival (RFS).12 We have also proposed that the intratumoural immune contexture  could be a new predictor of prognosis and provide a novel target for an optimal immunotherapy.13 As a result, the immune contexture was explored to tailor individual treatments and monitor the responses to anticancer therapies.9In situ immune cell infiltration in tumours is relevant for accurate prognostic predictions.14 In an international consortium led by the Society for Immunotherapy of Cancer, the densities of CD3+ and CD8+ T cells within the tumour and its invasive margin were summarised as Immunoscore, which showed the highest contribution of all clinical parameters to the recurrence risk and provided a relative estimate of the risk of recurrence in patients with colon cancer; thus Immunoscore was implemented as a new component of the tumour–nodes–metastasis (TNM)-Immune staging system.16 The Immunoscore classifier could effectively predict recurrence and survival in gastric cancer and was an ideal complementation to the TNM staging system.17 However, the characteristics of the HCC–immunohistochemistry (IHC) classifier are largely unknown.Currently, new data have revealed that Immunoscore, which is a classification based on the extent of tumour invasion by immune cells, provides a more accurate prognosis.Therefore, in this study, we used the least absolute shrinkage and selection operator (LASSO) Cox regression model to construct a novel HCC-IHC classifier to predict OS and RFS after surgery. The predictive performance of the HCC-IHC classifier was determined using time-dependent receiver operating characteristic (ROC) curves. Further, a nomogram that integrated the HCC-IHC classifier and four clinicopathological risk factors was established.The records of the patients with HCC who underwent primary tumour resection at Zhongshan Hospital, Fudan University  between 2005 and 2008 were reviewed. Their histopathological and clinical characteristics were scored according to the BCLC system Table . Two indAll haematoxylin and eosin-stained slides were examined by pathologists who were blinded to the clinical characteristics or outcome of the patient. Two cores were taken: one from the core of the tumour and the other from the peritumoural region as previously described. Tissue microarray construction was performed with a manual array instrument .20 ),22 24 and 26) in HCC. Detailed information is provided in + system and 3, 3’-diaminobenzidine-chromogen were applied to the slides  with 14 immune-related antibodies. In our study, 14 prognostic immune makers were chosen according to their close relationship with recurrence and survival ,27 The numbers of positive staining cells were recorded, and the mean value was used for statistical analysis. For each tumour, the three representative spots showed good level of homogeneity of stained cell numbers in each tumour and peritumour region.To evaluate the tissue-infiltrating immune cells, three most representative and independent fields were selected and captured at ×200 magnification. Identical settings were used for each photograph. The numbers of positive staining cells were counted using a computer-automated method  as described elsewhere.28The LASSO Cox analysis was adopted to select the most useful prognostic features out of all the HCC-associated immune features for predicting survival in the training cohort; R software version 3.0.1 and the “glmnet” package  were used to perform the LASSO Cox model analysis.t Test was used for continuous variables, and χ2 test was used to compare categorical variables between two groups. For the survival analysis, Kaplan–Meier method was used to analyse the correlations between variables and RFS, and log-rank test was adopted to compare survival curves. A Cox regression model was used to perform univariate and multivariate survival analyses, and nomograms were generated from Cox regression coefficients. The performance characteristics of the nomograms were explored by calibration plots. The clinical usefulness of the nomograms was evaluated by decision curve analysis (DCA). Nomograms and calibration plots were performed with the rms package of R software, and all the other statistical tests were performed with R software. The statistical significance level was set at 0.05.Table The median follow-up time was 52.2 months (interquartile range (IQR) 26.5–56.1), 53.2 months (IQR 35.3–57.1), and 42.4 months (IQR 30.8–46.3) in the training, testing, and validation cohorts, respectively. For the entire cohort, 353/664 (53.2%) patients developed tumour recurrence during the follow-up period, and 36.3% of the patients (241/664) died during follow-up. The 1-, 3-, and 5-year OS rates were 80.1%, 64.0%, and 59.2%, respectively, and the 1-, 3-, and 5-year RFS rates were 62.7%, 48.0%, and 39.6%, respectively.T , CD27T, CD4T, CD45ROT, CD27P , CD8P, CD4P, CXCR5T, CXCR5P, CD68P, CD45ROP, and CD45RAP, and the expression of CD45RA, CD45RO, and CXCR5 in tumour parenchyma were less than in peritumoural tissue  × 10–4. The level of each immune marker was measured as the number of stained cells positive for the specific immune marker in the tumour or peritumour tissue of the HCC tissue cores from the patient.IHC analysis of the samples from the 664 patients in the entire cohort showed a dominant cluster of CD8sue Fig. . X-tile 6bT Fig. . By usinP < 0.05 in the univariate analysis were enrolled into the multivariate analysis with the Cox regression model. Gamma-glutamyl transpeptidase (GGT) levels , 1.117–2.441; P = 0.012), liver cirrhosis , and the HCC-IHC classifier  were independent prognostic factors in the multivariate Cox model (Table P = 0.004) and validation cohort   Tables  and S3.Tχ2-value defined by Kaplan–Meier survival analysis and log-rank tests, the cut-off value was set at 0.148 with X-tile plots, ≥0.148 was considered as high, and <0.148 was considered as low in this formula.32 The distribution of the clinical characteristics did not vary significantly between the low-HCC-IHC classifier and the high-HCC-IHC classifier groups. The 5-year RFS rates were 46.7% for the low-HCC-IHC classifier group and 26.7% for the high-HCC-IHC classifier group in the training cohort . The calibration plot for the probability of survival at 3 or 5 years after hepatectomy showed optimal agreement between the prediction made by the nomogram and the actual observation , CLIP staging system (0.548), LCSGJ staging system (0.513), JIS staging system (0.508), Seventh TNM staging system (0.568), Eighth TNM staging system (0.568), and BCLC staging system (0.513)  Fig. .Fig. 4ROC-index of the HCC-IHC classifier nomogram for the prediction of RFS was 0.676, and a calibration curve presented good agreement between the predictions and observations for the probability of 5-year survival (Table C-index of the HCC-IHC classifier nomogram was higher than those of the other existing staging systems for the prediction of the RFS of HCC. The C-indices of the other systems were as follows: Okuda (0.560), CLIP (0.572), LCSGJ (0.530), JIS (0.530), Seventh TNM (0.559), Eighth TNM staging system (0.559), and BCLC staging system (0.530) , CLIP (0.591), LCSGJ (0.547), JIS (0.548), Seventh TNM (0.514), Eighth TNM staging system (0.569), and BCLC staging system (0.535). These results again suggested that the nomogram was useful for predicting survival in HCC  Fig. . In the al Table . The C-iHCC Fig. . In a DCHCC Fig. .C-index of HCC-IHC classifier was 0.649, which was higher than that of liver cirrhosis (0.568), tumour diameter (0.578), and MVI (0.552) and less than GGT (0.671). In the testing cohort, the C-index of HCC-IHC classifier was 0.615, which was higher than that of liver cirrhosis (0.556), tumour diameter (0.598), and MVI (0.583) and equalled to GGT (0.619). In the validation cohort, the C-index of HCC-IHC classifier was 0.590, which was higher than that of liver cirrhosis (0.532) and MVI (0.583), equalled to GGT (0.619), but less than that of tumour diameter (0.667) and MVI (0.602) , as in the training cohort, and we found that the 02) Fig. . These rC-index of HCC-IHC classifier was higher than other markers, and the results were reduplicated in the testing and validation cohort , a widely used statistic for the evaluation of co-linearity, of the selected features in the Cox model, and all the VIFs were <1.5, except one feature that had the VIF = 1.51 was not high to be considered that there was a co-linearity problem in our model.34 These systems have been broadly used in clinical practice but provide incomplete prognostic information. The outcomes after therapy for patients with HCC are variable even when patients are assigned to the same BCLC stage. In colorectal cancer (CRC), many new methods to classify cancer progression have been proposed; they mostly rely on tumour cell characteristics, such as morphology, molecular pathways, mutational burden, cell origin, and gene expression patterns.37 However, other major parameters, especially the tumour microenvironment, should also be taken into consideration. Thus the TNM-Immune staging system, which is a new classification, showed its superiority in predicting the prognosis of CRC treatment.39Until now, the staging of HCC has been largely dependent on histopathological criteria. Similar to the BCLC staging system, the Hong Kong staging system and HCC guidelines in China include tumour number, tumour diameter, and liver function.40 As an inflammatory modulator, TREM-1 correlated significantly with increased HCC recurrence and poorer survival.41 The activation status of tumour-infiltrating leukocytes was manipulated by the immunosuppressive gradient in primary HCC.42 Under the track of CCRL1, CCR7 (+) Treg-like cells facilitated tumour development and indicated adverse prognosis in HCC patients.43 In our study, the HCC-IHC classifier-based nomogram showed better accuracy in predicting both short- and long-term survival in the training, testing, and validation cohorts. We found that an increase in the HCC-IHC classifier was correlated with an adverse prognosis. As our result seems be in contrast with precious study which showed that high Immunoscore was associated with favourable prognosis, the underlying difference was that our HCC-IHC classifier was mainly composed of suppressive immune cell marker cluster, such as CD68, CD66b, and PD-1.45 Thus lower HCC-IHC classifier, which was in parallel with lower suppressive immune cell markers, is equal to hotter immune microenvironment or high Immunoscore, which was in line with more positive immune markers, which accentuated the finding in Galon and colleagues’ work.In HCC immunology study, OS and RFS were gradually prolonged as the Immunoscore increased.48 In HCC, the partial response rate for treatment with tremelimumab, which blocks CTLA-4, was 17.6%, and the disease control rate was 76.4%, which indicates that tremelimumab presents effective antitumour and antiviral activities.49 In another phase 1/2 dose escalation and expansion trial, the objective response rate was 20% in patients treated with 3 mg/kg nivolumab, which targets PD-1.50 Growing evidence supports Immunoscore as a prognostic biomarker for adjuvant therapy use. In gastric cancer treatment, adjuvant chemotherapy provided a better survival benefit to patients with stage II and III disease who were classified as high Immunoscore. However, the efficiency of the IHC classifier in HCC treatment guidance remains to be determined.Cancer immunotherapy is expanding rapidly due to the encouraging clinical results obtained with monoclonal antibodies that directly block checkpoint molecules, such as cytotoxic T-lymphocyte protein 4 (CTLA-4), PD-1, and its ligand PD-L1, that negatively regulate T cell responses.Our current study has limitations. First, it is a retrospective study with limited generalisability because all the patients were Chinese, and the clinical characteristics distribution might be more heterogeneous in other regions. Moreover, our study only enrolled BCLC stage 0 and A patients, making it susceptible to the inherent biases of such a study format. Second, although we assessed 14 immune markers, we could not encompass all the immune cell populations. New technological advances, such as single-cell transcriptome analysis and multiplex immunofluorescence assays, will improve the characterisation of tumour–immune interactions. Finally, our study was based on a single institution, and external validation is needed in our future work.In conclusion, our study established a novel standardised immune-based assay for the classification of HCC. The HCC-IHC classifier can effectively predict recurrence and survival and adds prognostic value to the BCLC staging system. A nomogram that includes the HCC-IHC classifier may help predict individual recurrence risks and help facilitate clinician decision-making for early-stage HCC patients.Supplementary Files


The results showed that Britanin treatment inhibited triple-negative breast cancer cell proliferation in vivo, and Cell Counting Kit-8  and colony formation tests (P < 0.001) confirmed this result. Transwell assays were conducted to verify that Britanin treatment inhibited cell migration and invasion (P < 0.001). Apoptosis was determined by TdT-mediated dUTP nick-end labeling method. Western blot and qRT-PCR analysis showed that Britanin treatment caused a decrease in the member expression of NF-kappa B signaling pathway. Computational modeling showed that Britanin could directly bind to a p-65 core region composed of Cys38, Cys120, and Gln128 residues. The results showed that the inhibitory mechanisms of Britanin on cancer cells may be by ways of inhibiting the NF-kappa B pathway. In addition, bioluminescence imaging screening system is useful for accelerating the application of Britanin in the antitumor field, and provides a useful tool for evaluating the phytochemicals efficacy in inhibiting cancer cell proliferation in animal models.Britanin has been reported to have therapeutic effects on neurodegenerative and inflammation-based diseases. However, whether it is involved in the regulation of triple-negative breast cancer development has not been elucidated. In this study, we investigated the anti-tumor activity against triple-negative breast cancer tumor of Britanin by bioluminescence imaging  Inula britannica. Previous studies have shown that Britanin inhibits the NF-κB pathway by inhibiting the degradation of IκB-α, nuclear translocation of NF-κB, and NF-κB/DNA binding activity  method. And the western blot and qRT-PCR method were performed to detect the expression of the proteins of related signaling pathways. A molecular docking simulation was performed to identify whether Britanin could bind to the NF-κB pathway protein p-65 via possible covalent binding sites.In this paper, the BLI method was used to assess whether Britanin has anti-breast cancer activity 2 in RPMI-1640 (GIBCO) supplemented with 10% fetal bovine serum , penicillin (100 IU/ml), and streptomycin (100 mg/ml). Cells were passaged three times a week. The Britanin working solutions (10 mM Britanin dissolved in DMSO) provided by Shanghai Jiaotong University were prepared by dilution of the stock solution in fresh culture medium on the day of use. Britanin, as a natural product, was purified by high-performance liquid chromatography and characterized by nuclear magnetic resonance (NMR) spectroscopy. The purity of Britanin was greater than 95%  were incubated at 37°C with 5% CO4 cells/ml) were seeded in 96-well plates (100 μl) at 37°C overnight with 5% CO2 and then incubated with various concentrations of Britanin  at 37°C for 72 h. Four wells containing only complete medium were used as blank control group, and four wells containing tumor cells suspended in the complete medium which were used as control group. Thiazolyl blue tetrazolium bromide  was added to each well. After incubation for 4 h, using a Multiskan Ascent microplate photometer, the absorbance was measured at 492 nm wavelength. The control group cells were regarded as having a 100% survival rate. And the percentage of growth inhibition was calculated as cell growth inhibition (%) = (treated OD-blank OD)/(control OD-blank OD) × 100%. The concentration required for a 50% inhibition of viability (IC50) was then determined.Measurements of cell viability at different drug concentrations were performed. MDA-MB-231 cells, MDA-MB-231 luc cells, SUM-159 cells, and SUM-159 luc cells (1×10MDA-MB-231 luc and SUM-159 luc cells (500 cells/well) were seeded in 12-well flat-bottomed plates and incubated for 24 h. Using the complete medium as the control group and different concentrations of Britanin as the experimental group, MDA-MB-231 luc cells and SUM-159 luc cells were tested. Cells were pretreated with different concentrations  of Britanin for 48 h. The treatment medium was replaced with normal growth medium and was replaced with normal growth medium every 3 d; After 2 weeks of incubation, formed colonies were fixed with 4% paraformaldehyde for 10 min, stained with 0.5% crystal violet for 10 min, washed again with PBS and photographed. The results were quantified with Adobe Photoshop Software.5 cells/ml. Then, Britanin with different concentrations  were added to the cells. Four wells containing the solution served as the control group. Simultaneously, 500 μl of 10% FBS-supplemented medium was added to the lower chamber. Cells were removed from the upper Matrigel chamber membrane using a cotton swab after 24 h of incubation and were attached to a glass slide, fixed with methanol and stained with Giemsa. Three fields per chamber were analyzed by light microscopy for migrated cell quantification. The experiment was repeated three times.For the Transwell migration experiment, cells were seeded into the upper Matrigel filled chamber of a 24-well Transwell plate with 200 μl of serum-free medium. The MDA-MB-231 and SUM-159 cells density were adjusted to 2×106 cells/site), Britanin was intraperitoneally injected. Model mice were randomly divided into two or three groups . Britanin dissolved in PBS. Mice were treated once every other day. The size of the tumor in vivo and the weight of mice were measured and compared with that of the xenograft tumors in mice of the placebo control. BLI was recorded during the experiment. D-luciferin  was injected with the mice 10 min before BLI. The animals were then anesthetized with 2% isoflurane and 0.3 L/min of oxygen. The area of regions of interest (ROIs) image processing and analysis visualized using Live Image 4.5 software. To quantitative analysis, the ROIs of the tumor were evaluated with a white light image, and muscle regions of similar size (opposite positioning with the tumor) were selected as muscle ROI. The background value is subtracted from each of the luminescence images. The average of the fluorescence signal within each ROI is then calculated. Simultaneously, tumor volume was also calculated in vitro using calipers. After the mice were sacrificed, tumor volume was calculated with calipers, tumors and organs were fixed with 10% formalin solution.All animal studies carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources and approved by the University of Xi'an Jiaotong Animal Care and Use Committee (number XY-AUC-2017-213). Two weeks after the implantation of cancer cells  in PBS for 30 min and then immersed in 4% paraformaldehyde for 5 min at room temperature. The slides were rinsed twice with PBS for 5 min per rinse. Then, the slides were equilibrated with 100 μl of equilibration buffer at room temperature for 5 min, and the slides were incubated with 100 μl of rTdT reaction mix per slide at 37°C in a humidified chamber for 1 h. Next, the slides were immersed in 2×SSC at room temperature for 15 min. Each slide was incubated with 100 μl of streptavidin HRP solution for 30 min and washed twice in PBS for 5 min per wash at room temperature. The slides were washed twice again with PBS for 5 min per wash at room temperature. At the last step, diaminobenzidine peroxidase substrate was added, and the slides were analyzed by light microscopy. The excised tumors were also stained with hematoxylin-eosin (H&E).The cells were cultured at 0 and 5 mg Britanin for 24 h respectively. The cells were subsequently lysed using buffer with protease inhibitors. Total protein (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) and transferred to polyvinylidenedifluoride membranes. Membranes were blocked with 5% fat-free milk/TBST and incubated with primary antibodies and secondary antibodies. Antibodies against p-65, p150/p50, p-p65, and housekeeping gene, GAPDH were obtained from Abcam. Secondary antibodies were HRP-conjugated antibodies and visualized by a chemiluminescent reagent , p50F: 5′-AGTAGCTGAGAGGCACATGG-3′, p50R: 5′-AGCGCACTCCAACCTTCTCA-3′, oligonucleotide sequence of p65 (256 bp), p65F: 5′-GCACTTACGGATTCTGGTGG-3′, p65R: 5′-GCACTTACGGATTCTGGTGG-3′, oligonucleotide sequence of GAPDH (252 bp), GAPDHF: 5′-CACTGGCATGGCCTTCCGTG-3′, GAPDHR: 5′-GAAATGAGCTTGACAAAGTG-3′. The quantification of each sample of cDNA was performed in triplicate, each PCR was replicated three times for verification, and the 2In this study, the monomer in the p-65 was used as the initial structure and receptor protein (PDB coded: 1IKN) . Cys38 oData are presented as the mean values ± SD of independently repeated experiments, with values of P < 0.05 considered to represent a statistically significant difference between compared data.50) of Britanin in MDA-MB-231 luc, MDA-MB-231, SUM 159 luc, and SUM 159 cell lines were 4.27, 4.41, 5.05, and 5.33 μM, respectively. Luc-labeled cells and non-Luc-labeled cells showed the same reactions to Britanin, so Luc-labeled cells were used in subsequent experiments.As indicated in in vitro.As shown in As shown in in vivo. Initially, 1×105 cells were implanted into the bilateral legs of mice. At 2 weeks post-implantation, when the luminescence signal of cells reached 2×105 p/s/cm2/sr, the mice were injected with Britanin once every 2 d or the placebo. By the 14th day post-treatment, the growth of the luminescence signal of MDA-MB-231 luc cells implanted in mice treated with Britanin  was smaller than that of MDA-MB-231 luc tumors in the mice of the placebo control  was studied by BLI continuously in vivo bioluminescence signal of the test group tumor was significantly lower than that of the placebo control group and suggested that the growth rate of tumors was inhibited by Britanin  into BALB/c mice xenotransplant model of SUM-159 luc cells or MDA-MB-231 luc cells, the major organs  were harvested for histopathological analysis. No noticeable tissue or cellular damage was observed in the H&E-stained organ slices from Britanin-treated mice compared to those from nude mice treated with saline as control, except for some binuclear nucleolar cells in the liver , and the optimal energy conformation was selected by analyzing the results of 100 docking iterations. Ligands enter the target protein active sites mainly through hydrophobic and van der Waals chemical forces and interact with amino acid residues, including Lys37, Cys38, Gln89, Gln119, Cys120, Val121, Lys122, Asp125, Gln128, and Ala129. The docking energy is −6.07, and the binding activity of the ligand to the target protein is noncovalent. Oxygen atoms in the five-membered ring of the ligand form hydrogen bonds with Gln128, and the hydrogen bond length is 3.1 Å. The formation of hydrogen bonds is beneficial to small molecule-targeting proteins, thus inhibiting p-65 activity  is defined as a subtype of breast cancer and has the features of aggressive behavior, poor prognosis and no standard chemotherapy protocols. A comparison of TNBC with normal breast tissue showed substantial changes in the NF-κB signaling pathway that controls angiogenesis and tumorigenesis. Thus, NF-κB p-65 is a common and effective therapeutic drug target. Many plant-derived compounds inhibit the NF-κB pathway in breast cancer, such as triptolide parthenolide, hirsutine, and andrographolide. Britanin is a guaiacyl-type sesquiterpene lactone and an inhibitor of the NF-κB pathway that controls inflammatory responses. Therefore, we evaluated the anti-TNBC activity of Britanin using athymic nu/nu mice implanted with MDA-MB-231 luc and SUM-159 luc cells expressing a luciferase reporter gene in vivo BLI process, the luciferase report gene was transfected and used to label both of the above cell types. Cell lines stably expressing luciferase were established. The BLI in vivo results showed that there were no cell-specific effects by this method. The luminescence intensities of ROIs after treatment with Britanin were examined from the bioluminescence images continuously. And the trend in these results were similar with the trend in the tumor volume growth values. The experimental results show that there is a difference between tumor volume measured in vivo and tumor volume dissected. Because the tumor sizes of the experimental group and control group are different, their accurate measurement may be easily influenced by subjective factors. It is worth noting that by the traditional method the difference in tumor size between the test group and control group of mice was not evident on the 9th day and could not be observed until after the 14th day. However, from the first observation point (4th day or 9th day), the ROI value of the experimental group increased less than that of the control group. Meanwhile, real-time monitoring of BLI adds information to the effects of different Britanin concentrations and enables consecutive observation. Noninvasive examination in vivo combined with conventional and classical methods. The metabolically active cells in the BLI response, especially in small tumors, can be accurately detected, avoiding the impact of factors such as peritumoral edema for the traditional tumor volume measurement. Thus, Bioluminescence assays are more sensitive than traditional methods for detecting tumor size in vivo and vitro.In our study, the MDA-MB-231 cells derived from the pleural effusions of a Caucasian breast cancer patient and SUM-159 cells derived from a noninflammatory metaplastic patient selected for this screen were used to elucidate whether Britanin inhibits cell growth. In the The TUNEL analysis revealed that compared to the control treatment, Britanin treatment markedly induced apoptosis in MDA-MB-231 and SUM-159 cells. The results herein indicate that Britanin markedly inhibits cell proliferation in MDA-MB-231 and SUM-159 human TNCB cancer cells in a dose- and time-dependent manner. The western blot and qRT-PCR analysis results showed that the expression of p-p65 of the test group cells was lower than that of the control group, and the total expression of p-65 has no difference between the test group and the control group. And NF-κB p-65 and p-50 mRNA levels had less than 10% increase. NF-κB is a dimeric form of transcription factor family ubiquitous in eukaryotes. In the cytoplasm, the NF-κB subunits p-65 and p-50 exist as heterodimers and form a complex with its inhibitory protein IκB. This complex in the cytoplasm covered the nuclear localization signal of the p-65, so the complex was in an inactive state. The p-65 was phosphorylated to exert its transcriptional effect. In addition, Britanin contains sesquiterpenoid lactones with α-methylene-γ-butyrolactone moiety. The binding mode prediction is that Michael-type thiols bind to cysteine sulfhydryl groups of p-65.via the inhibition of the NF-κB pathways. Another conclusion is that this bioluminescence evaluation method allows the simultaneous visualization of changes in tumor volume and could be applied to new types of drug screening, discovery, and development.In conclusion, the results of the present study show a novel use of Britanin in mitigating human TNBC proliferation and inducing apoptosis The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.All animal studies carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources and approved by the University of Xi'an Jiaotong Animal Care and Use Committee (number XY-AUC-2017-213).XX carried out biological experiments. YG carried out animal experiments. GD and HL analyzed data and review the manuscript. LW and DC wrote the paper and led the research.This work was supported, in part, by the National Key R&D Program of China under Grant No. 2018YFC0910600 and the Natural Science Basic Research Plan in Shaanxi Province of China under Grant No. 2019JQ-519 and 2018JM7072.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Allium sativum L.), which was heated for various lengths of time, using X-band (9 GHz) electron paramagnetic resonance (EPR) and high performance liquid chromatography (HPLC) were investigated. Non-spin-trap and non-destructive EPR detected the total reaction intermediates . The g-value of the signal was 2.004. The signal with a peak-to-peak linewidth (ΔHpp) was approximately 0.67 milli Tesla (mT). The values of the intermediates are suggestive of organic compounds. The garlic darkened in color with the increasing number of heating days. Melanoidin, responsible for darkening of the garlic, was detected at an absorbance of 400 nm. Analysis of the correlation between the EPR intensity and melanoidin absorbance showed a good correlation coefficient (0.98). In addition, 5-hydroxymethyl furfural (5-HMF) and total phenolic compounds increased with the increasing number of heating days. Moreover, trace amount of Fe3+ was observed in the black garlic by EPR. Non-destructive EPR is a useful method for evaluating not only Maillard reaction intermediates, but also the pigment associated with the reaction processes.The Maillard reaction intermediates and related phytochemicals in garlic ( Allium sativum L. garlic, came into popularity in Asian countries  and 0.1% formic acid acetonitrile [solvent B]. The linear gradient elution was performed as follows: time t (min), solvent A (%): , , , and , with a flow rate of 0.65 mL/min and an injection volume of 1.0 µL. UV−visible (UV/vis) absorption spectra were recorded from 200 to 600 nm using a photodiode-array detector (PDA). The wavelength for UV detection was set at 280 nm. The 5-HMF content was calculated using a standard curve prepared with a 5-HMF . The limit of detection (LOD) was 0.3125 µg, the minimum limit of quantification (LOQ) was 0.625 µg.The HPLC-ESI(electrospray ionization)-Tof(time-of flight)-MS analysis was performed with a Xevo G2-XS QTof . The chromatographic conditions were the same. The HPLC ESI-TOF-MS spectra were acquired by scanning from m/z 100 to 1000 with a capillary voltage of 3.0 kV, cone voltage of 30 eV, and source temperature of 150 °C. Nitrogen was used as a nebulizing gas at a flow rate of 20 L/h. The 5-HMF reagent was purchased from Tokyo Chemical Industry Co., Ltd. . p < 0.05. Analyses were conducted using commercial software . Results are expressed as mean ± standard deviation (SD) or standard error (SE). Statistical analyses between multiple groups were conducted using analysis of variance. Statistical comparisons were made using Tukey‒Kramer tests. Differences were inferred as significant for After heating the garlic, the color of garlic after seven days of heating changed to light brown . The gar3+. The g-value for Fe-ions can vary depending on the moieties of the compound. Note that oxidation state of iron may have been changed during heating the sample, since there are no detectable iron ions in the untreated garlic spectrum. The concentration of free minerals was reported in a previous study [us study . The signal at around 336.0 mT (g ~ 2.00) likely corresponds to intermediate radicals ,16. Sinc17. The number of heating days and the sample weights are indicated on the left-hand and right-hand sides of the chart, respectively. Four different garlic samples for each heating day were measured. EPR intensity is generally proportional to the amount (weight) of the samples measured. The amount of each garlic sample was not exactly the same when we prepared the garlic samples. Hence, the intensity was normalized, and an average of all measurements was considered. The EPR signal intensities increased with the number of heating days. In addition, the results can be inferred from a relation between dark color of the sample and EPR intensity (ntensity .+ was 127.0393 (m/z) and the molecular weight was expected to be 126.11  (e 126.11 . Based o97 (m/z) . Further97 (m/z) . These r97 (m/z) .It has been previously reported that black garlic has a higher polyphenol content than raw garlic . The polWith regard to polyphenol and its intermediate, a previous study suggested that the stability of phenoxyl radical intermediate was indicated in the antioxidant sesamol . Thus, wThe X-band EPR detected reaction intermediates in the various garlic samples, which increased in concentration upon increasing number of days of heating. The present analyses suggested that melanoidin, 5-HMF, and phenolic compounds are produced with various periods of garlic heating. EPR intensity may be a good index of garlic browning. Thus, the EPR and HPLC are very useful techniques that can be applied in the evaluation of Maillard reaction in foodstuffs.


Beckmannia syzigachne Steud.) is one of the most troublesome weeds infesting wheat and canola fields in China. Some biotypes cannot be controlled, either by acetolactate synthase (ALS) or acetyl coenzyme A carboxylase (ACCase) inhibitors, which are the main herbicides for controlling this weed. However, very few studies have investigated multiple resistance mechanism in B. syzigachne. In this study, a B. syzigachne biotype with a high resistance to ALS inhibitors we have reported was also showed relatively lower resistance to ACCase inhibitors, with a resistance index around 7. RNA-seq analysis was used to investigate the factors responsible for multiple resistance, and 60,108 unigenes were assembled by de novo transcriptome assembly and then annotated across eight databases. A Pro-197-Ser mutation was identified in the ALS gene by SNPs analysis and validated by PCR, while no mutation was identified in the ACCase gene. Nineteen candidate metabolic genes were screened and their overexpression was confirmed by qPCR. The expression of GST-T3 and GST-U6 in resistant plants ranged from 7.5- to 109.4-folds than that in susceptible ones at different times after two kinds of herbicide treatment. In addition, GST activities in resistant plants were 3.0–5.0 times higher than that in susceptible plants. Other novel resistance factors also showed high correlation with multiple resistance which included four genes encoding disease resistance proteins, a transcription factor (MYC3), and one gene conferring blight resistance. In this research, a B. syzigachne biotype was confirmed to have evolved multiple resistance to ACCase and ALS inhibitors. The Pro-197-Ser mutation in ALS gene and high-level GST activities were confirmed responsible for the multiple resistance. Characterized disease-resistance proteins, transcription factor, and blight-resistance proteins may play an essential role in these multiple herbicide resistance.American sloughgrass ( Beckmannia syzigachne Steud.) is a troublesome diploid weed (2n = 14), which threatens many crop varieties in China such as wheat (Triticum aestivum L.) and oilseed rape [Brassica rapa L. subsp. Oleifera (DC.) Metzg.]  and non-target-site resistance (NTSR) . TSR is patterns . With reerbicide . NTSR iserbicide . To dateerbicide . Many geerbicide .B. syzigachne to ACCase-inhibiting herbicides mainly involves TSR, in which amino acid substitutions have been identified at six positions  in resistant B. syzigachne  explore the multiple resistance factors using next generation sequencing methods, (3) confirm the Single Nucleotide Polymorphisms (SNPs) or the expression for the candidate genes, and (4) determine the enzyme activity which relative to the multiple resistance.Hence, this research aimed to (1) determine the multiple resistance levels of B. syzigachne biotype were collected from Wuxi city (WC1148) and found evolved high-level resistance to ALS inhibitors in our previous study (® XR8002 flat fan nozzle under a pressure of 0.275 MPa (Resistant (R) us study . A susceus study . Three k.275 MPa . Dry wei-1 of fenoxaprop-P-ethyl and the 20 g a.i. ha-1 mesosulfuron-methyl, respectively. After 24 h, the leaf tissues of three biological replicates were random collected, and also the samples without herbicide treatment were collected as controls with three individuals for each biotype. All the 18 samples were snap frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted using the RNAprep Pure Plan Kit . The degradation and contamination were checked by 1.0% agarose gel electrophoresis. RNA purity and concentration were determined using the NanoDrop-1000 spectrophotometer . The RNA integrity was assessed by the Agilent Bio analyzer 2,100 system   .de novo transcriptome assembly was carried out using the Trinity platform and the longest transcript of each locus was termed a “unigene” for subsequent annotation (-5), BLAST2GO, and Blastall were used to predict and classify the different unigenes, as in previous studies  . Paired-notation . Eight d studies .Brachypodium distachyon [L.] Beauv. The software GATK2 was used for SNP discovery and the detection stringency conditions include at least five reads calling the variant and >30 mapping quality score  using the Hi-DNAsecure Plant Kit (DP350) by following the manufacturer’s instructions . Two pairs of PCR primers were design to clone ALS ; the amplification products of these two pairs of primers were 1035 and 766 bp in size, respectively, which encompass all of the reported mutation sites. One pair of primers was designed  to clone ACCase containing the CT region, and the PCR products were 1308 bp in size. The PCR mixture components and amplification conditions were in accordance with those reported in a previous study . The experiment of PCR was done twice and had 15 biological replicates both for R and S biotypes.For SNP calling, picard tools was used to map the paired-reads from each sample to the reference genome sequence of ty score . To confus study , and theP values in multiple tests and analyses , and magnitude of differences in expression between the above treatment groups (|log2 (fold change)| ≥ 1). quantitative Real-time PCR (qPCR) was performed to confirm the accuracy of their expression. The best reference gene was selected from among capsine phosphatase (CAP), glyceraldehyde 3-phosphate dehydrogenase (CADP), and ubiquitin (UBQ) by the software BestKeeper  were detected using the qPCR method for all the samples, respectively, and the GST activity for the samples were also detected using a Glutathione S-transferase Microplate Assay Kit (CAK 1047) following the manufacturer’s instructions . Each sample (0.1 g leaf tissues) was assayed at 340 nm using a Tecan Infinite 200 Pro plate reader . The results were expressed as U g-1, and one unit of GST activity was defined as the enzyme generating 1 μmol of glutathione and 1-chloro-2, 4-dinitrobenzene conjugate in the reaction time. Both the gene expression and GST activity assay were repeated twice and at least three biological replicates were performed for each treatment.The plant culture for the R and S biotypes, and the herbicide treatment were same as described above. At 0, 24, 48, and 72 h, leaf samples for each treatment (at least three replicates) were collected and stored at -80°C. Total RNA was exacted and first strand cDNA synthesis was performed using the methods described above for all samples. The expression levels of two GSTs-related genes (50) were estimated through nonlinear regression with a log-logistic model:The values of herbicide doses required to reduce 50% of the plant growth . The difference of gene expression and GST activity between R and S biotypes were analyzed by the Student’s t-test, which were performed with SPSS 13.0 .using Sigma Plot 12.0  . In this50 values of the S biotype WC1004 were 42.4, 12.1, 11.4, and 11.8 g a. i. ha-1, respectively. For the R biotype WC1148, the GR50 values were 87.0, 39.4, 84.5, and 89.2 g a. i. ha-1, respectively. Compared with those of the sensitive biotypes, the RI of WC1148 were 2.1, 3.3, 7.4 and 7.5, respectively (The sensitivity of the R (WC1148) and S (WC1004) biotypes in response to four different ACCase inhibitors  was calculated. The GR260/OD280 values (around 2.0) and the RNA integrity number (RIN) values (>6.0) indicated that the RNA integrity of the samples was suitable for the requirements of subsequent experiments. The cDNA library of 18 RNA samples were analyzed: 449,583,465 raw reads, and 436,188,739 clean reads which ranged from 22,724,569 to 24,516,824 per sample were generated, respectively  of the total 118,111 unigenes were successfully annotated in at least one database, and 8,197 unigenes were annotated in all of the database (ALS gene (c38811_g1) using the RNA-seq analysis, while, no SNPs was found for the ACCase gene  which causing the amino acid Pro change to Ser  to serine TCC (Ser), and no other mutation sites were found in any of the 15 individuals | ≥ 1 and q < 0.05) were selected as candidate resistance genes. The number of up- and down-regulated genes in CK1148 vs CK1004 combination was 700 and 907, respectively. For the group set of M1148 vs CK1148, and F1148 vs CK1148, the number of up- and down-regulated genes was no more than 300. After herbicide treatment, differentially expressed genes between the R and S biotypes (M1148 vs. M1004 and F1148 vs. F1004) were more than 400. The number of up- and down-regulated genes in the M1004 vs. CK1004 combination was 2,720 and 4,185, respectively. However, the number of up- and down-regulated genes in the F1004 vs. CK1004 combination was 162 and 233, respectively  annotation to GSTs were analyzed at different times after herbicide treatment. The results show that expression of these two genes in the R individuals was higher than that in the S individuals at each time point under herbicide treatment  were also found after fenoxaprop-P-ethyl treatment. Without herbicide treatment, the expression of GST-U6 was 46.5 times higher than in S and show slight decreased after these two kinds herbicide treatment.The expression levels of two unigenes , which was 5.0 times higher than that in S biotype. However, it was slightly decreased in the S biotype, which is around 0.8 U g-1. Similar results were also found in these two biotypes after fenoxaprop-P-ethyl treatment. The GST activities were increased in R biotype and can reach to 4.9 U g-1, while GST activities in the S biotype was around 1.0 U g-1 at all the time points. GST activities in the R plants were significantly higher than those in the S plants at all the time points.To determine whether the higher expression of these genes of GSTs increase the GST active in R biotype. GST activity was investigated in both R and S biotypes at 0, 24, 48, and 72 h after mesosulfuron-methyl and fenoxaprop-P-ethyl treatment. The results show that in R plants without herbicide treatment, the GST activity was 3.4 U gB. syzigachne biotype had evolved multiple resistance to ALS and ACCase inhibitor herbicides. In our previous research, this biotype showed higher resistance to mesosulfuron-methyl, and also exhibited higher cross-resistance to other ALS inhibitor herbicides, such as flucarbazone, imazapic, pyroxsulam, and pyribenzoxim  Schrad  was found among the biotypes resistant to ALS inhibitor  Schrad . For B. nhibitor . To dateBeckmannia syzigachne has evolved resistance to ACCase inhibitors, and many genes involved in NTSR were discovered using the RNA-seq technology (GST-T3 and GST-U6) were found to be overexpressed at different times after mesosulfuron-methly and fenoxaprop-p-ethyl treatment in resistant B. syzigachne. What is more, the GST activity in resistant plants higher than the susceptible ones at all the detected times. This result is similar to that reported in a previous study  and regulatory genes  . Beckmanchnology . Metabolchnology  Esterasechnology . In thisus study . This coB. syzigachne, some new genes related to resistance were also found in this research. Four genes annotated as encoding disease resistance proteins  in the resistant population were expressed at 65.8-1124.0 times higher levels than that in the sensitive biotype. This phenomenon was found both before and after herbicide treatment. This suggested that the disease-resistance protein might be related to the resistance. The expression levels of oxidase (L-pipecolate oxidase and iron/ascorbate family oxidoreductases) in R biotypes were 10.0–103.4 times higher than those in sensitive biotypes after treatment, which may be related to fenoxaprop-p-ethyl metabolism in individuals.In addition to the reported resistance factors in In this study, besides disease-resistant proteins, transcription factors and other regulatory genes were also screened. Protein kinases play key roles in signal transduction pathways, bringing external signals into cells, amplifying cascades of reactions, and passing them to transcription factor proteins to regulate the expression of downstream functional genes. Plant immune receptor is often classified as threonine kinase, and studies have shown that tyrosine phosphorylation in plants has an important regulatory role in innate immunity . MYC2 haB. syzigachne biotype that had evolved multiple resistance to ALS and ACCase inhibitor herbicides, and which showed high resistance to ALS inhibitor and relatively lower resistance to the ACCase ones. A Pro-197-Ser mutation was found in the ALS gene and no mutations were found in the ACCase gene. Four genes encoding disease resistance proteins, the MYC3 transcription factor, and one gene for blight resistance protein were found related to confer multiple resistance. Of the novel resistance factors, two GST genes (GST-T3 and GST-U6) showed high correlation to this multiple resistance. The results of this research could provide the important resistant factors about multiple resistance to ACCase and ALS-inhibiting herbicide in B. syzigachne.In this research, we found a The raw Illumina sequence reads have been deposited in the NCBI Sequence Read Archive (SRA) database. The accession numbers for the samples with herbicide treatment were SRR10737896 and SRR10737895. And the accession numbers for the samples without herbicide treatment were SRR10742660 and SRR10742659.This research was designed by HC. JW and JC performed the experimental work and the data analysis. XL, HC, DL, ZL, and JC provided helpful suggestions for the data analysis and manuscript revision. JC wrote the manuscript. All authors contributed to the article and approved the submitted version.This research was funded by The National Natural Science Foundation of China (no. 31371952).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.